Golden Rice & Golden Crops

Transcription

1 Golden Rice & Golden Crops Transgenic Plants for Food Security in the Context of Development Peter Beyer University of Freiburg Germany Pontifical Academy of Science, Vatican, May 2009

2 Nutritional Diversity Iron, Zinc Folate Provit A Vit. E Rice (grain) Tomato - - (+) + Beans Spinach

3 Nutritional Diversity Iron, Zinc Folate Provit A Vit. E Facts: Rice Three billion - live on - less than - 2 $ per day, billion on less than 1 $ per day and cannot afford a Tomato diversified diet or - industrially - produced (+) supplements + Beans Spinach Meat + + Vit A + - Millions are chronically micronutrient malnourished

4 Intervention strategies: Supplementation Industrial fortification Education All necessary and very valuable but there are drawbacks: Distrubution, educated medical staff Centrally processed food items Only partially applicable Economically sustainable?

5 Biofortification is an alternative to classical interventions to fight micronutrient deficiencies Improve the nutritional value of agronomically important crop tissues through 1. Breeding 2. Recombinant DNA technologies is all achievable through breeding? Simple answer: NO!!

6 1. Some crop plants do not show adequate trait variability Rice, (polished grains) for instance Provitamin A: Germplasm screening did not reveal any yellow grains with β-carotene Folate: Practically absent Iron: low variability, ranging from 1 8 ppm (Final breeding target 14 ppm) Zinc: much more important variability, ranging from ppm (Target: 24 ppm)

7 Golden Rice cannot be bred.the application of recombinant DNA technology is necessary

8 Why engineering ß-carotene (provitamin A) biosynthesis into rice endosperm? Milled rice is provitamin-a-free Symptoms of a provitamin-a-free diet Night-blindness Xerophthalmia Fatal susceptibility to childhood diseases (e.g. measles) and general infections (diarrhoea, respiratory diseases) Epidemiology 124 million children are deficient in vitamin A 1-2 million deaths annually (1-4 years) million deaths (5-10 years) UNICEF; Humphrey et al., 1992) A severe public health problem in (118) countries (WHO)

9 Xerophthalmia

10 Why engineering ß-carotene (provitamin A) biosynthesis into rice endosperm? Milled rice is provitamin-a-free Symptoms of a provitamin-a-free diet Night-blindness Xerophthalmia Fatal susceptibility to childhood diseases (e.g. measles) and general infections (diarrhoea, respiratory diseases) Epidemiology 124 million children are deficient in vitamin A 1-2 million deaths annually (1-4 years) million deaths (5-10 years) UNICEF; Humphrey et al., 1992) A severe public health problem in (118) countries (WHO)

11 HOW? Assembly-line technologies E8 E7 E6 E5 E4E3 E2 E1 Precursor Intermediates Expressed genes DNA, mrna Product

12 (HOW?) E1 Starting Point: Wild-type IPP PP PP DMAPP PP E2 GGPP-Synthase GGPP E3 Phytoene-Synthase Phytoene Phytofluene E4 Phytoene Desaturase ζ-carotene Neurosporene Lycopene E5 E6 E7 E8 (Cis/trans Isomerase?) ζ-carotene Desaturase Lycopene cis/trans Isomerase α, β-lycopene Cyclase β-carotene α-carotene All missing (not expressed) in rice endosperm???

13 (HOW?) E1 Towards a concept: PSY (E3) transformation IPP Wild-type rice endosperm PP PP DMAPP PP E2 GGPP-Synthase GGPP E3 Phytoene-Synthase Phytoene Phytofluene E4 Phytoene Desaturase ζ-carotene E5 (Cis/trans Isomerase?) Neurosporene E6 ζ-carotene Desaturase Lycopene E7 Lycopene cis/trans Isomerase E8 α, β-lycopene Cyclase β-carotene α-carotene Wild-type endosperm can produce a precursor molecule, GGPP!

14 Assembly-line technologies E8 E7 E6 E5 E4E3 E2 E1 Precursor Intermediate Only two appeared at work! Product

15 (HOW?) E1 PP PP IPP Wild-type rice endosperm DMAPP PP E2 GGPP-Synthase GGPP E3 Phytoene-Synthase Phytoene Phytofluene E4 Phytoene Desaturase ζ-carotene E5 (Cis/trans Isomerase?) Neurosporene Lycopene E6 E7 E8 ζ-carotene Desaturase Lycopene cis/trans Isomerase α, β-lycopene Cyclase β-carotene α-carotene It is a nightmare to transform six transgenes. Luckily there is CrtI!

16 Luckily there is CrtI transformed E. coli Pantoea ananatis carotenoid gene cluster crte crtx crty crti crtb crtz ORF12 ORF2 ORF3 ORF4 ORF6 CrtI substitutes for 4 plant genes

17 Carotene Desaturases Complex vs. simple Cyanobacteria and Plants Bacteria 15-cis-Phytoene 15-cis-Phytoen E4 PDS E5 E6 Ζ-ISO?? ZDS 9, 15, 9 -tri-cis-z-carotene 9, 9 -di-cis-z-carotene CRTI E7 CRTISO 7, 9, 9, 7 -tetra-cis-lycopene all-trans-lycopene all-trans-lycopin

18 The CrtI gene product provides a shortcut B Plant Desaturation pathway CrtI shortcut A

19 (HOW?) E1 Towards a prototype: The concept IPP Wild-type rice endosperm PP PP DMAPP PP E2 GGPP-Synthase E3 GGPP E3 Phytoene-Synthase Phytoene Phytofluene E4 Phytoene Desaturase CrtI ζ-carotene E5 (Cis/trans Isomerase?) Neurosporene E6 ζ-carotene Desaturase E7 Lycopene E7 E8 Lycopene cis/trans Isomerase α, β-lycopene Cyclase β-carotene α-carotene Three instead of six!!

20 Pathway Complementation in Rice, Co-Transformation (1) pzpsc Gt1p E3 (PSY) 35Sp E,4,5,6,7 (tp-crti) (2) pzlcyh aph IV 35Sp E8(ß-LCY) Gt1p With the selectable marker gene only in (2), all yellow transgenic seeds expressed lycopene-ß-cyclase. They all contained ß-carotene; this was the expected outcome.

21 Control

22 hpc11

23 But there was a second construct without lycopene cyclase! aph IV Gt1p 35Sp pb19hpc E3 (PSY) E3,4,5,6,7 (tp-crti) Single transformant hpc 2b Lutein Zeaxanthin α-carotene ß-Carotene Lesson learned: no need for lycopene ß-cyclase why is Golden Rice golden (yellow) instead of red??? Ye et al., 2000; Science 287:303

24 E1 We just need to bridge a gap!! Wild-type rice endosperm IPP PP PP DMAPP PP E2 GGPP-Synthase E3 GGPP E3 Phytoene-Synthase Phytoene Phytofluene E4 Phytoene Desaturase CrtI ζ-carotene E5 (Cis/trans Isomerase?) Neurosporene E6 ζ-carotene Desaturase Lycopene E7 Lycopene cis/trans Isomerase E8 α, β-lycopene Cyclase β-carotene Xanthophylls α-carotene Only two transgenes are necessary!! Schaub et al. (2005), Plant Physiol. 138: 441

25 Assembly-line technologies E8 E7 E6 E5 E4E3 E2 E1 Precursor Intermediate Only two transgenes are necessary to fill the gap!! Product

26 Prototypes: Not apt for product development Construct ill-defined Integration ill-defined Antibioticselectable marker Low amount of bc (1.6 µg/g) Happy Easter Start from scratch include Indica rice varieties.

27 Improved Golden Rice variants came in two versions In the public and in the private sector (Syngenta - Orynova) Gt1p PSY (Np) Gt1p tp-crti (from Narcissus) No selectable marker gene (co-transformed and removed) Almost 1000 events Deregulation-amenable integration CrtI controlled by an endosperm-specific promoter In Cocodrie (Javanica) Amount up to 6 µg/g Three events preselected Known as Golden Rice 1 Technology works in Indica varieties

28 A B T 2 Rice grains C D E F G H Hoa et al., Plant Physiol. 133, 2003

29 The preselected events (PS&S) underwent 2 field trials at Louisiana State University

30 where the GR1 events showed 4,8 7,1 µg/g

31 Improvements: The past years were dominated by efforts to increase the amount of ß-carotene in GR both, in the public sector as well as at Syngenta

32 Potential bottlenecks to higher carotenoid levels C3-Carbon Metabolism IPP/DMAPP Phytoene synthase (E3) activity? GGDP Phytoene Desaturation ζ-carotene (CrtI) activity? Lycopene Precursor shortage? Inefficient transgene transgene expression? expression? Carotenoid storage? ß-Carotene Zeaxanthin α-carotene Lutein

33 Improving CrtI (E4,5,6) expression PMI 35Sp PSY (Np) pfun3 promoter change & codon optimized pcarnew promoter change GluBp Synth tp crti + control Western WT CarNew E1-19 (T1) CarNew E4-4 (T1) Cacar (T3) Cacar (T3) Achieved!!! But no significantly improved ß-carotene accumulation. Carotene desaturation is not rate-limiting in Golden Rice CrtI PSY

34 Phytoene synthase was investigated by Rachel Drake (Syngenta) Because PSY expression is good in GR, different versions of the PSY gene were assayed. Daffodil Psy Seed promoter CrtI Seed promoter Ubi promoter hyg R Maize Psy Rice Psy Transformation into a japonica short-grain rice, (Asanohikare) 20+ plants each Tomato Psy Pepper Psy Carotenoid content (µg g -1 dwt) rice Psy/crtI maize Psy/crtI pepper Psy/crtI tomato Psy/crtI Individual transgenic plant (event) Rice and Maize PSY (E3) Best. Proportion of ß-carotene increased. daffodil Psy/crtI

35 Assembly-line technologies E7 OK CrtI OK OK CrtI CrtI Too slow!!! E1 E3 E2 Precursor Zwischenprodukt Produkt

36 Golden Rice 2 was made for implementation GT1pI tp-crti GT1pI ZmPSY ubi1p PMI psyn12424 Transform long grain rice variety (Kaybonnet) Sugar selectable marker 619 individual GM rice plants Screen for seed colour, gene copy number, fertility Select 6 Golden Rice 2 events for further screening and development

37 Improved provitamin A Accumulation in Golden Rice I and II

38 GR 2 Contains the bacterial CrtI and and PSY (but from maize) just like the previous versions. Both genes are under endosperm specific promoter control; the selectable marker agent is mannose. Increase in provitamin A content is about 10-fold over GR1 and about 25-fold over the prototype Golden Rice is mainly a breeding project today: Philippines (IRRI, PhilRice) Vietnam (CLDRI) India (IARI,TNAU, DRRI) Bangladesh (BRRI) Introgressing 8 events into 11 varieties Event selection completed, moving towards deregulation

39 GR2 GR1 Wild-Type

40 1. Some crop plants do not show adequate trait variability Maize, for instance Maize is the world s third most important staple crop. In maize, the pathway proceeds beyond beta carotene. The genetic variability for high carotenoid levels is very substantial, but low for provitamin A carotenoids. A Psy-CrtI combination, as used in GR boosts ß-carotene production in an African white cultivar to 60 µg/g! Breeding approaches (ongoing - lyce polymorphisms identified) have yielded so far ca.14 µg/g ß-carotene. Naqvi et al., PNAS, 2009 Harjes et al., Science

41 2. Some crop plants show adequate trait Variability but cannot be (easily) bred Bananas, for instance Bananas are a staple in 50 (+) countries (Uganda; 222 kg/person year) East Africa Highland Bananas are very low in micronutrients (ProvitA 2.7 µg/g; Vit E 1 µg/g; Iron 2.6 ppm, fresh weight) Conventional breeding: extremely difficult as bananas are essentially sterile Most current cultivars are sterile triploids selected from the wild Have not been genetically improved for thousands of years Huge challenges from global movement of devastating diseases James Dale, QUT Australia, Grand Challenges in Global Health

42 Transient testing of the transgenes using direct Agrotransformation of banana fruits NT Ubi-CrtI Ubi-Apsy2a Ubi-Apsy2a+CrtI Preliminary HPLC data indicated increased α-carotene, β-carotene and lutein

43

44 2. Some crop plants cannot be (easily) bred Cassava, for instance 250 million sub-saharan Africans and 600 millions globally rely on cassava as their major source of calories Ranks 5 th among crops directly consumed by humans (No. 1 in Sub-Saharan Africa). Provides food security. Very low in micronutrients Provit A (mostly) 1-5 µg/g; VitE, 1 µg/g; Iron 5 ppm, Zinc 1 ppm (fresh weight) Varietal recovery very difficult upon breeding (vegatatively propageted) Very long breeding cycle Richard Sayre, Danforth Center, St. Louis, USA, Grand Challenges in Global Health, BMGF University of Freiburg CIAT, Harvest Plus

45 Cassava promoter CP2 - crtb Line #12 more lines epressing multiple genes coming this year

46 2. Some crop plants cannot be (easily) bred Potato, for instance potato ranks fourth, among the staple foods of mankind, after wheat, rice and maize pk-i 35S TP CrtI Nos pk-bi Pat1 TP CrtB Ocs 35S TP CrtI Nos pk-ybi Nos CrtY TP 35S CrtB CrtI Pat1 TP Ocs 35S TP Nos pp-i Pat2 TP CrtI Nos pp-bi CrtB CrtI Pat1 TP Ocs Pat2 TP Nos pp-ybi Pat2 CrtY TP CrtY Nos CrtB CrtI Pat1 TP Ocs Pat2 TP Nos only this one: A mini-pathway

47 Diretto et al., PlosOne, 2007 Golden Potato

48 All of the here-mentioned examples bear significant potential: They represent the major staples The provitamin A bioavailability is very good! Bioavailability: Golden Rice: 3.8:1, Human (maybe even better) Tang et al., Am J Clin Nutr 2009 Maize: 3:1 Gerbil, human study is underway Howe and Tanumihardjo, J. Nutr Cassava: 3.7:1, Gerbil Howe et al., British Journal of Nutrition (2009) Spinach: 20:1, Human Tang et al., Am J Clin Nutr 2005; Very good bioavailability of ß-carotene from simple starchy food matrices (Like banana and potato?)

49 The problem of VAD remains : GoldenRice and other Golden Crops are a potentially significant contribution to alleviation. Genetic modification is an indispensible tool Breeding where possible Genetic modification where necessary

50 To all our sponsors since before 1990: ETH / Swiss Federal Funds European Commission HarvestPlus USAID Syngenta Company Syngenta Foundation National Institutes of Health (USA) Bill and Melinda Gates Foundation The Golden Rice Humanitarian Board Bayer, Mogen, Novartis, Monsanto, Orynova, Zeneca Rice Teams & IRRI Phil Rice- Philippines CLRRI- Vietnam DBT, IARI, DRR,TNAU- India

51 On our way we meet... Suspicion

52 ...Mimicry

53 strange places

54 strong opposition... GoldenRice Field Trial (one week before harvest!) Hurricane Ivan September 2004 Hurricane Katrina August 2005 Hurricane Rita September 2005 Typhoon Fengshen June 2008

55

56 Promoter Gene Current status CP crtb Field Patatin crtb-crti-crty* Field, Greenhouse Extensin Yam Sugar beet crtb crtb-crti-crty crtb crtb-crti-crty crtb crtb-crti-crty Transformed cell lines Transformed cell lines Under construction Transformed cell lines

57 GLOBAL AREA OF BIOTECH CROPS Million Hectares (1996 to 2007) Total Industrial Developing 23 Biotech Crop Countries Increase of 12%, 12.3 million hectares (30 million acres), between 2006 and Source: Clive James, 2007.

58

59

60 Developing countries have already benefited from modern agricultural biotechnology products Performance Advantage of Insect Resistant over Conventional Cotton (expressed as a percentage) Argentina China India Mexico S. Africa Yield Revenue Pesticide costs Seed costs Profit Based on peer-reviewed studies of 2-3 seasons of commercial farm production Adapted from: Raney. T. (2006) Economic impact of transgenic crops in developing countries. Current Opinion in Biotechnology 17:1 5